Fungal sepsis, or fungemia, is a serious public health concern afflicting over 100,000 patients every year in the US where almost 38% of those do not survive. In fungemia early targeted treatment is life-saving, and considering the rate at which the disease develops, is perhaps the most crucial factor to improving outcomes. Unfortunately, given the time-consuming diagnostic process today, the underlying cause of this disease is typically identified too late. Current diagnostics do not facilitate early initiation of a targeted antifungal intervention - as they require culturing, which requires as long as 2-5 days for accurate identification of the infecting pathogen. Hence current treatment protocols use 'best-guess' approaches which are not only less effective, but likewise increase complications, and increase the prevalence of drug resistant pathogens. Having met our and exceeded our Phase I Specific Aims, this Phase II proposal focuses on the automation of the entire diagnostic into a fully-integrated, sample-to-answer consumable enabling the automated species- level identification of the most clinically prevalent fungal pathogens directly from phlebotomy specimens, in roughly 2 hours; without human intervention. The sample-to-answer diagnostic we will develop will have profound impact, both by improving outcomes by providing a means to develop a hypothesis driven first-line intervention, and likewise by reducing the use of excessive and unnecessary antimicrobials. To succeed in this Phase II we have put together a top-notch team at HelixBind, including experts in assay development, application of synthetic nucleic acid analogues, the design and implementation of sample-to- answer automated diagnostics, and in biomedical instrumentation development. Additionally we have enlisted key strategic advisors in clinical microbiology, surface chemistry, and nucleic acid analogues as well as successful entrepreneurs with experience in the development and commercialization of medical devices. Together, we will build upon our already impressive Phase I results, having established species level capability at sub-10 CFU/ml sensitivity levels directly from spiked human samples. To establish the performance capability our automated and culture-free diagnostic, we will culminate this Phase II by assessing the clinical specificity and sensitivity of our diagnostic wit the assistance of our clinical partner. Having achieved our Phase II Specific Aims, we will move forward and develop deployable instrumentation/consumables and validate our entire diagnostic in preparation for the pivotal clinical trial.